1. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
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Research Article CODEN: IJRPJK ISSN: 2319 – 9563
ANALYTICAL TECHNIQUES (CHROMATOGRAPHY, SPECTROSCOPY,
ELECTROPHOROSIS) IN PHARMACEUTICAL ANALYSIS: A REVIEW
Imran Khan*1
, Krantisri Mulpuri2
, Byasabhusan Das1
, MD Mohiuddin1
, Mohd Haseeb Ur Rahman3
*1
Department of Quality Assurance, Anwar Ul Uloom College of Pharmacy, Hyderabad, India.
2
Department of Quality Assurance, Emergent Labs, Hyderabad, India.
3
Department of Chemistry, Northeastern Illinois University, Chicago, USA.
INTRODUCTION
The process of drug development starts with the
innovation of a drug molecule that has showed
therapeutic value to battle, control, check or cure
diseases. The synthesis and characterization of such
molecules which are also called active
pharmaceutical ingredients (APIs) and their analysis
to create preliminary safety and therapeutic efficacy
data are prerequisites to identify the drug candidates
for further detailed investigations.
ABSTRACT
The development of the pharmaceuticals brought a revolution in human health. These pharmaceuticals would
serve their intent only if they are free from impurities and are administered in an appropriate amount. To make
drugs serve their purpose various chemical and instrumental methods were developed at regular intervals
which are involved in the estimation of drugs. These pharmaceuticals may develop impurities at various stages
of their development, transportation and storage which makes the pharmaceutical risky to be administered,
thus they must be detected and quantitated. For this analytical instrumentation and methods play an important
role. This review highlights the role of the analytical instrumentation and the analytical methods in assessing
the quality of the drugs. The review highlights analytical techniques such as chromatography, spectroscopy,
electrophoresis and their corresponding methods that have been applied in the analysis of pharmaceuticals.
KEY WORDS
Analytical Instruments, Pharmaceuticals, Quality, Chromatography, Spectroscopy and Electrophoresis.
International Journal of Research
in
Pharmaceutical and Nano Sciences
Journal homepage: www.ijrpns.com
Author for correspondence:
Imran Khan,
Department of Quality Assurance,
Anwar Ul Uloom College of Pharmacy,
Hyderabad, Telangana State, India.
Email: Imrankhan503015@gmail.com.

2. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 20
In the field of pharmaceutical research, the
analytical investigation of bulk drug materials,
intermediates, drug products, drug formulations,
impurities, degradation products, and biological
samples containing the drugs and their metabolites is
very important. From the commencement of official
pharmaceutical analysis, analytical assay methods
were included in the compendial monographs with
the aim to characterize the quality of bulk drug
materials by setting limits of their active ingredient
content. In recent years, the assay methods in the
monographs include titrimetry, spectrometry,
chromatography, and capillary electrophoresis; also
the electro analytical methods can be seen in the
literature.
From the stages of drug development to marketing
and post marketing, analytical techniques play a
great role, be it understanding the physical and
chemical stability of the drug, impact on the
selection and design of the dosage form, assessing
the stability of the drug molecules, quantitation of
the impurities and identification of those impurities
which are above the established threshold essential
to evaluate the toxicity profiles of these impurities to
distinguish these from that of the API, when
applicable and assessing the content of drug in the
marketed products. The analysis of drug and its
metabolite which may be either quantitative or
qualitative is extensively applied in the
pharmacokinetic studies.
This review highlights the role of various analytical
techniques and their corresponding analytical
methods in the analysis of pharmaceuticals.
ANALYTICAL TECHNIQUES
Chromatographic Techniques
Chromatography is an analytical technique based on
the separation of molecules due to differences in
their structure and/or composition. In general,
chromatography involves moving a sample through
the system over a stationary phase. The molecules in
the sample will have different affinities and
interactions with the stationary support, leading to
separation of molecules. Sample components that
display stronger interactions with the stationary
phase will move more slowly through the column
than components with weaker interactions. Different
compounds can be separated from each other as they
move through the column1
.
THIN LAYER CHROMATOGRAPHY
In thin layer chromatography, a solid phase
(stationary phase), the adsorbent, is coated onto a
solid support as a thin layer (about 0.25 mm thick).
In many cases, a small amount of a binder such as
plaster of Paris is mixed with the absorbent to
facilitate the coating. Many different solid supports
are employed, including thin sheets of glass, plastic,
and aluminum. The mixture (A plus B) to be
separated is dissolved in a solvent and the resulting
solution is spotted onto the thin layer plate near the
bottom. A solvent, or mixture of solvents, called the
eluents, is allowed to flow up the plate by capillary
action. At all times, the solid will adsorb a certain
fraction of each component of the mixture and the
remainder will be in solution. Any one molecule will
spend part of the time sitting still on the adsorbent
with the remainder moving up the plate with the
solvent. A substance that is strongly adsorbed (say,
A) will have a greater fraction of its molecules
adsorbed at any one time, and thus any one molecule
of A will spend more time sitting still and less time
moving. In contrast, a weakly adsorbed substance
(B) will have a smaller fraction of its molecules
adsorbed at any one time, and hence any one
molecule of B will spend less time sitting and more
time moving. Thus, the more weakly a substance is
adsorbed, the farther up the plate it will move. The
more strongly a substance is adsorbed, the closer it
will stay near the origin.
HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
HPLC: A Powerful Separation Method
A powerful separation method must be able to
resolve mixtures with a large number of similar
analytes. A chromatogram provides directly both
qualitative and quantitative information: each
compound in the mixture has its own elution time
(the point at which the signal appears on the screen)
under a given set of conditions; and both the area

3. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 21
and height of each signal are proportional to the
amount of the corresponding substance. This
example shows that high-performance liquid
chromatography (HPLC) is very efficient, i.e. it
yields excellent separations in a short time. The
‘inventors’ of modern chromatography, Martin and
Synge were aware as far back as 1941 that, in
theory, the stationary phase requires very small
particles and hence a high pressure is essential for
forcing the mobile phase through the column. As a
result, HPLC was sometimes referred to as high-
pressure liquid chromatography3
.
SPECTROSCOPIC TECHNIQUES
Spectrophotometry
Another important group of methods which find an
important place in pharmacopoeias are
spectrophotometric methods based on natural UV
absorption and chemical reactions.
Spectrophotometry is the quantitative measurement
of the reflection or transmission properties of a
material as a function of wavelength.
The advantages of these methods are low time and
labor consumption. The precision of these methods
is also excellent. The use of UV-Vis
spectrophotometry especially applied in the analysis
of pharmaceutical dosage form has increased rapidly
over the last few years4
.
The Light of Knowledge is an often used phrase, but
it is particularly appropriate in reference to
spectroscopy. Most of what we know about the
structure of atoms and molecules comes from
studying their interaction with light (electromagnetic
radiation). Different regions of the electromagnetic
spectrum provide different kinds of information as a
result of such interactions.
Mass Spectrometry
Sample molecules are ionized by high energy
electrons. The mass to charge ratio of these ions is
measured very accurately by electrostatic
acceleration and magnetic field fluctuations,
providing a precise molecular weight. Ion
fragmentation patterns may be related to the
structure of the molecular ion.
Ultraviolet-Visible Spectroscopy
Absorption of this relatively high-energy light
causes excitation of electrons. The easily accessible
part of this region (wavelengths of 200 to 800 nm)
shows absorption only if conjugated pi-electron
systems are present.
Infrared Spectroscopy
Absorption of this lower energy radiation causes
vibrational and rotational excitation of groups of
atoms within the molecule. Because of their
characteristic absorptions identification of functional
groups is easily accomplished.
Nuclear Magnetic Resonance Spectroscopy
Absorption in the low-energy radio-frequency part
of the spectrum causes excitation of nuclear spin
states. NMR spectrometers are tuned to certain
nuclei (e.g. 1
H, 13
C, 19
F and 31
P). For a given type of
nucleus, high-resolution spectroscopy distinguishes
and counts atoms in different locations in the
molecule5
.
HPLC Coupling With Various Spectroscopic
Techniques
HPLC can be combined with numerous other
analytical techniques but the most important
coupling principle is the one with spectroscopy.
Chromatography and spectroscopy are orthogonal
techniques, i.e. their types of information are very
different. Chromatography is a separation method
and spectroscopy is a technique which yields a
‘fingerprint’ of molecules. Coupling with atomic
spectrometry is rarely used although it allows the
detection of toxic metals in environmental samples
or of metalloproteins6
. Four other techniques,
HPLC-UV, HPLC-FTIR, HPLC-MS and HPLC-
NMR are more important because excellent spectra
are obtained with them, thus allowing structure
elucidation.
HPLC-UV: The Diode Array Detector7
Compared to conventional UV detectors, the diode
array detector is built with inverse optics. Figure
No.1 shows the principle of this instrument. The full
light first goes through the detector cell and is
subsequently divided spectrally in a polychromator
(which is a grating). The spectral light then reaches
the diode array, a chip with a large.

4. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 22
Number (100-1000) of light-sensitive diodes that are
arranged side by side. Each diode only obtains a
well defined fraction of the information which is
read by the electronics for data processing. This type
of detector allows a wealth of information to be
obtained from UV spectra. The most important
possibilities are presented in Figure No.2:
• It is possible to obtain and store spectra of
individual peaks during a chromatogram. This
process does not take more than half a second,
including data processing; therefore it is possible
to obtain several spectra, even from narrow
peaks. For identification the spectra can be
compared on-line with a library. In order to
improve correlations, the computer calculates the
second derivative which shows more maxima
and minima than the original spectrum.
• The knowledge of spectra of the compounds
involved then allows detection at selected
wavelengths. Interfering peaks can be eliminated
due to the proper choice of detection conditions.
It is often possible to obtain an accurate
quantitation of a compound even if the
resolution in principle is poor. The detection
wavelength can be altered during a
chromatographic run4
.
• Simultaneous detection at two different
wavelengths allows calculation of the
absorbance ratio8
. If this ratio is constant over
the whole width of a peak it can be assumed that
it is pure (with the exception of the exact co-
incidence of two peaks with identical shape or of
an impurity with identical UV spectrum). If the
ratio is not constant this is proof that the peak is
not pure4
.
• Subtraction of two wavelengths allows baseline
drift to be reduced during gradient elution as
well as noise. The reference wavelength can be
chosen in a region where none of the compounds
of interest shows absorption.
HPLC-FTIR9
The coupling with Fourier-transform infrared
spectroscopy allows spectra to be obtained. The
detection limit is rather high (i.e. poor) if a flow-
through cell is used. The interfaces with solvent
elimination are more interesting and flexible but
technically more demanding; they can even register
spectra of trace analytes. A volatile mobile phase is
needed; it may be aqueous.
HPLC-MS10
Mass spectrometers for HPLC consist of three
different parts: the interface where the eluate enters
the MS and the ions are generated, the mass
analyzer, and the detector, an electron multiplier
which determines the ion beam intensity. Thanks to
the different possibilities for ion generation at
atmospheric pressure, HPLC-MS is possible with
rather low expenditure.
The main two ionization techniques are APCI and
ESI.
APCI: Atmospheric pressure chemical ionization
(Figure No.3).
• Ions are generated by corona discharge (3-6 kV).
• Yields molecule ions (M + H) +
(no spectra),
negative ionization is also possible.
• Suitable for small, medium and nonpolar
molecules; however, they need some proton
affinity and volatility.
• Not suitable for thermolabile analytes,
• For aqueous and nonaqueous mobile phases,
flow must be at least 1 ml min-1
. With
nonaqueous eluents no additives are necessary;
reactions with the solvent are possible during
ionization.
• With aqueous eluents an additive may be
necessary for efficient ionization.
• Mass-sensitive signal.
ESI or APESI: (Atmospheric pressure)
electrospray ionization (Figure No.4).
• Ions are generated by ‘coulomb explosion’
(disintegration) of electrically charged droplets.
• Yields ions with single or multiple charge; in the
latter case, spectra with many peaks are obtained
which must not be mixed up with classical
spectra showing molecule fragments.
• Suitable for thermolabile analytes and
macromolecules, including biopolymers.
• Suitable for aqueous eluents; flow must be small,
therefore useful for micro HPLC.

5. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 23
• For positive ionization a pH of 5 is suitable,
additives are formic and acetic acid, perhaps
together with ammonium acetate:
Analyte+H AnalyteH+
+A-
•
For negative ionization a pH of 9 is suitable,
additives are ammonia, triethylamine and
diethylamine, perhaps together with ammonium
acetate:
AnalyteH+B Analyte-
+HB+
Concentration-sensitive signal.
Quadrupoles and ion traps are the most frequently
used mass analyzers. Both systems are rather low-
priced and robust but their resolution is limited to
1Da. The ions can be sent through several mass
analysers and fragmentations, setup in series, thus
yielding more information about the structure. These
techniques are known as MS/MS (two steps) or MSn
(n steps).
HPLC-NMR11
Nuclear magnetic resonance can be coupled with
HPLC if the solvents are chosen properly (one
solvent signal can be suppressed but the others must
be avoided by using deuterated solvents) and if the
analyte concentration is not too low. Detection limit
depends on radiofrequency; therefore a 500-
800MHz instrument must be used. If the analyte
concentration is high enough, it is possible to run
online HPLC-NMR separations, but usually the
individual peaks are stored in loops and spectra are
obtained offline with a long enough measuring
time4
.
Comparison between High-Performance Liquid
Chromatography and Gas Chromatography
Like HPLC, gas chromatography12
(GC) is also a
high-performance method, the most important
difference between the two being that GC can only
cope with substances that are volatile or can be
evaporated intact at elevated temperatures or from
which volatile derivatives can be reliably obtained.
Only about 20% of known organic compounds can
be analysed by gas chromatography without prior
treatment. For liquid chromatography, the sample
must be dissolved in a solvent and, apart from cross-
linked, high-molecular-mass substances; all organic
and ionic inorganic products satisfy this condition.
The characteristics of the two methods are compared
in Table No.2. In comparison with gas
chromatography there are three important
differences:
• The diffusion coefficient of the sample in the
mobile phase is much smaller in HPLC than in
GC. (This is a drawback because the diffusion
coefficient is the most important factor which
determines the speed of chromatographic
analysis).
• The viscosity of the mobile phase is higher in
HPLC than in GC. (This is a drawback because
high viscosity results in small diffusion
coefficients and in high flow resistance of the
mobile phase).
• The compressibility of the mobile phase under
pressure is negligibly small in HPLC whereas it
is not in GC. (This is an advantage because as a
result the flow velocity of the mobile phase is
constant over the whole length of the column.
Therefore optimum chromatographic conditions
exist everywhere if the flow velocity is chosen
correctly. Moreover, incompressibility means
that a liquid under high pressure is not
dangerous).
Comparison between High-Performance Liquid
Chromatography and Capillary Electrophoresis
Capillary electrophoresis13
(also termed capillary
zone electrophoresis, CZE) is suited for electrically
charged analytes and separates them, simply
speaking, according to their ratio of charge to size.
In addition, the shape of the molecules is another
parameter which influences their speed; therefore
the separation of isomers or of analytes with
identical specific charge is possible. Cations
(positively charged molecules) move faster than
anions (negatively charged molecules) and appear
earlier in the detector. Small, multiply charged
cations are the fastest species whereas small,
multiply charged anions are the slowest ones.
The separation is performed at high voltage. An
electric field of up to 30 kV is applied between the
ends of the separation capillary. As a consequence,
the buffer solution within the capillary moves
towards the negatively charged cathode. The

6. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 24
capillaries have a length of 20-100 cm and an inner
diameter of 50-250 mm. In contrast to HPLC they
are not packed with a stationary phase in the
chromatographic sense but in some cases with a gel
which allows the separation of the analytes by their
size (as in size-exclusion chromatography).
The separation performance can be of much higher
order of magnitude than in HPLC (up to 107
theoretical plates), making CE an extremely
valuable method for peptide mapping or DNA
sequencing. However, small molecules such as
amino acids or inorganic ions can be separated as
well. The absolute sample amounts which can be
injected are low due to the small volume of the
capillaries. A major drawback is the lower
repeatability (precision) compared to quantitative
HPLC. Preparative separations are not possible.
Electrokinetic chromatography is a hybrid of HPLC
and CE. For this technique the capillaries are packed
with a stationary phase and the separation is based
on partition phenomena. The mobile phase acts as in
CE; it consists of a buffer solution and moves thanks
to the applied electrical field4
.
Table No.1: Lists a number of adsorbents in the order of adsorptive power2
Most Strong Adsorbent
Alumina
Charcoal
Florisil
Al2O3
C
MgO/SiO2 (Anhydrous)
Least Strong Adsorbent Silica Gel SiO2
Table No.2: Comparison of GC and HPLC4
S.No Problem GC HPLC
1 Difficult separation Possible Possible
2 Speed Yes Yes
3 Automation Possible Possible
4
Adaptation of system to
separation problem
By change in stationary
phase
By change in stationary
and mobile phase
5 Application restricted by
Lack of volatility, thermal
decomposition
Insolubility
6 Typical number of separation plates Per column Per metre
7 GC with packed columns 2000 1000
8 GC with capillary columns 50 000 3000
9 Classical liquid Chromatography 100 200
10 HPLC 5000 50 000
Figure No.1: Principle of a diode array detector4

7. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 25
Figure No.2: Possibilities of the diode array detector. (a) Registration of spectra during the separation;
(b) registration of a chromatogram at different wavelengths; (c) peak purity control; (d) chromatogram
with and without subtraction of a reference wavelength4
Figure No.3: HPLC-MS interface for APCI. 1¼ from HPLC column; 2¼ nebulizing gas; 3¼ heater; 4¼
corona discharge needle; 5¼ drying gas; 6¼ skimmer; 7¼ quadrupole.4

8. Imran Khan. et al. / International Journal of Research in Pharmaceutical and Nano Sciences. 4(1), 2015, 19- 27.
Available online: www.uptodateresearchpublication.com January – February 26
Figure No.4: HPLC-MS interface for ESI. 1¼from HPLC column; 2¼ nebulizing gas; 3¼ high voltage;
4¼ charged droplet (drawn too large); 5¼evaporation; 6¼ coulomb explosion; 7¼ drying gas; 8¼
skimmer; 9¼quadrupole.4
CONCLUSION
Chromatography and spectroscopy are orthogonal
techniques, i.e. their types of information are very
different. Chromatography is a separation method
and spectroscopy is a technique which yields a
‘fingerprint’ of molecules. HPLC is a technique for
separation, identification and quantification of
components in a mixture. It is especially suitable for
compounds which are not easily volatilized,
thermally unstable and have high molecular weights.
But whereas GC is effectively used for analysis of
gas mixtures or liquids having low boiling points.
On the other hand liquid chromatography is applied
for separation of thermally labile liquid mixtures or
those having high boiling points.
The advantage of UV method over HPLC method is
that the UV method does not require the elaborate
treatment and procedures usually associated with
chromatographic method. It is less time consuming
and economical .The HPLC and UV spectrotometry
methods are adequate methods to quantify a drug in
pure form and its dosage form. Since these methods
are simple, specific, rapid, precise and accurate, they
may be successfully and conveniently adopted for
routine quality control analysis of drugs in bulk and
pharmaceutical dosage form.
A major drawback of CE is the lower repeatability
(precision) compared to quantitative HPLC.
ACKNOWLEDGEMENT
The authors wish to express their sincere gratitude to
Anwar Ul Uloom College of Pharmacy, Hyderabad,
India for providing necessary facilities to carry out
this research work.
CONFLICT OF INTEREST
We declare that we have no conflict of interest.
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Please cite this article in press as: Imran Khan et al. Analytical Techniques (Chromatography,
Spectroscopy, Electrophorosis) In Pharmaceutical Analysis: A Review, International Journal of Research
in Pharmaceutical and Nano Sciences, 4(1), 2015, 19 - 27.